Leukemia stem cells (LSCs) are regarded as the origins and key therapeutic targets of leukemia, but limited knowledge is available on the key determinants of LSC “stemness”. Using single cell RNAseq analysis, we identify a master regulator PU.1, whose LSC-specific expression determines the molecular signature and activity of LSC in the Pten-null T-ALL model. Although initiated by PTEN controlled β-catenin activation, PU.1 expression and LSC “stemness” are maintained by a β-catenin-PU.1-TIM-3 feedback loop, independent of the leukemogenic driver mutation. Perturbing any component of this loop, either genetically or pharmacologically, can prevent LSC formation or eradicate existing LSCs. LSCs lose their “stemness” when PU.1 expression is silenced by DNA methylation, which can be reactivated by 5-AZ. Importantly similar regulatory mechanisms are also present in human T-ALLs. Overall design: Here we performed Bulk RNA-seq analysis for LSCs and Blast cells from BM, thymus and spleen in PTEN null T-ALL mouse model.